Zebrafish (Danio rerio) has emerged as ?Drosophila of the vertebrates? to study genetic controls of development and disease. It has several advantages over mice due to its small size, large progeny, and short reproduction cycle. Transparent embryos and ex vivo development allows noninvasive observations of embryonic development. Furthermore, the ability to do saturation mutagenesis using ENU (N-ethyl-N-nitrosourea) followed by forward and reverse genetic technologies to isolate mutants has a great potential in functional genomics. We have undertaken a chemical mutagenesis screen using ENU in the zebrafish to uncover mutants in early hematopoiesis. Haploid embryos from F1 females were screened by RNA in situ hybridization using a stem cell marker, core binding factor b (cbfb), and a myeloid-specific marker, leukocyte-specific plastin (l-plastin). We have screened 510 F1 females and 47 putative mutants have been identified. Of these, 22 showed decreased expression of l-plastin, 3 for cbfb, 2 for both l-plastin and cbfb and the remainder were mutated for l-plastin and other non-hematopoietic markers. Of the lines with defect in the expression of cbfb, we recovered 2 mutants. The first mutant line, L32, displayed a bloodless phenotype and showed a significant decrease in the expression of the hemangioblast marker scl. In addition to the marked reduction in the expression of the myeloid specific markers l-plastin, c/ebp1 and mpo, these embryos also showed decreased expression of the erythroid specific marker gata-1. Linkage analysis has placed this mutant to the proximal region of LG 12. Currently, we are in the process of testing candidate genes to identify the mutated gene. The second mutant line, L63 also has a bloodless phenotype and showed decreased expression of both l-plastin and mpo. Like L32, L63 homozygous embryos have significant developmental defects and die 3 dpf. Low resolution mapping has placed this mutant to LG 8. Through this approach, we hope to identify novel genes that play critical roles in the developmental process regulating early hematopoiesis. We have also undertaken an effort to generate a mutant library archived as a sperm bank and a DNA bank from ENU-mutagenized F1 males, in order to screen for mutations in specific genes of interest. DNA was used to screen for mutations by sequencing or other heteroduplex screening methods. The corresponding sperm samples were used to revive the mutant fish by in vitro fertilization. In last year we archived sperm and DNA from 1300 F1 male fish and we have been in the process of screening for mutations in a selected set of genes. These were 12 genes involved in hematopoiesis, cancer, development, and signal transduction. We have identified 7 mutations affecting 5 of these genes even though we have not completed all the sequencing and data analysis. We have derived lines for 2 of these mutations by IVF. These data support the feasibility of the approach. We are also in the process of generating additional mutants to increase the size of the library, thereby giving us a better chance to identify mutations in any given gene. Finally, we are generating transgenic fish expressing green fluorescent protein gene and leukemia fusion genes under the control of regulatory sequences from several genes expressed in blood cells. Such transgenic fish will be useful tools for our genetic screens and for the generation of leukemia models.